Between the staple and unique (<i><font color="red">CTGTCGCATCGAGAG</font></i>) sequences, 15 T bases are inserted. They are to make a T loop. Thanks to this T loop, single-stranded DNA complementary to the unique sequences (such as Anchored DNA) are expected to easily hybridize with the unique sequence.<br>

+

Between the staple and unique (<i><font color="red">CTGTCGCATCGAGAG</font></i>) sequences, 15 T bases are inserted. They are to make a T loop. Thanks to this T loop, single-stranded DNA complementary to the unique sequences (such as Origami-anchor DNA) are expected to easily hybridize with the unique sequence.<br>

The 30nt single-stranded parts are stable till 37 degrees, according to <A Href="http://www.nupack.org/">NUPACK</A>).<br>

The 30nt single-stranded parts are stable till 37 degrees, according to <A Href="http://www.nupack.org/">NUPACK</A>).<br>

The 141 staples have the same length so that they may be present at the same intervals in the DNA origami.<br>

The 141 staples have the same length so that they may be present at the same intervals in the DNA origami.<br>

Line 139:

Line 139:

<br>

<br>

<h6>The list of strands</h6>

<h6>The list of strands</h6>

-

The other strands exept DNA origami staples used in our experiment are shown in Table1.<br>

+

The other strands exept DNA origami staples used in our experiment are shown in Table2.<br>

-

The sequence of cholesterol-conjugated DNA (in the rest of this document, referred to as Anchored DNA) is shown below (at the first sequence in Table1). For labeling, we also attached fluorescent tagged DNA (at the second in Table1) to our DNA origami.<br>

+

The sequence of Origami-anchor DNA is shown below (at the first sequence in Table2). For labeling, we also attached fluorescent-tagged DNA (at the second in Table2) to our DNA origami.<br>

-

To hybridize different strands of Anchored DNA and fluorescent tagged DNA with the same unique single-stranded parts of our origami, we arranged two kinds of adaptor DNA (at the third and fourth in Table1). One adaptor has complementary sequences to both the unique sequence and Anchored DNA. The other has complementary sequences to both the unique sequence and the fluorescent tagged DNA. Thanks to these two adaptors, two different strands can bind to the same unique sequence. <br>

+

To hybridize both Origami-anchor DNA and fluorescent-tagged DNA with the same unique single-stranded parts of our Origami, we arranged two kinds of adaptor DNA (at the third and fourth in Table2). One adaptor has complementary sequences to both the unique sequence and Origami-anchor DNA. The other has complementary sequences to both the unique sequence and the fluorescent-tagged DNA. Thanks to these two adaptors, two different strands can bind to the same unique sequence. <br>

Then we added 2µl DNA origami into each sample and saw if some change would happen with a fluorescent microscope.<br>

Then we added 2µl DNA origami into each sample and saw if some change would happen with a fluorescent microscope.<br>

-

The DNA origami for fluorescent microscope observation was made according to Table3 annealing solution. It contained more cholesterol-hybridizing ssDNAs and fluorescent-tagged DNA-hybridizing ssDNAs than Annealing solution used in 1-1), because we considered a sample with more fluorescent molecules was suitable for observation. <br>

+

The DNA origami for fluorescent microscope observation was made according to Table4 annealing solution. It contained more cholesterol-hybridizing ssDNAs and fluorescent-tagged DNA-hybridizing ssDNAs than Annealing solution used in 2-1-1, because we considered a sample with more fluorescent molecules was suitable for observation. <br>

1 Step1 Disruption of temperature sensitive liposomes

1-1 Disruption of temperature sensitive liposomes

Structure of NIPAM

poly-N-isopropyl acrylamide

Making liposome

Egg York PC(10mM)

10µl

Cholesterol(10mM)

1µl

CHCl3

90µl

TXR

1µl

Table1 Materials for Making liposomes

1. Drying the liposomes above with argon gas and letting them stand for a night
2. Adding L paraffin 100µl to 1 and sonicating them for an hour
3. Picking up 10µl from 2, adding 25μl NIPAM2mg/ml to them and vibrating them with Vortex

2-1 DNA Origami approach

2-1-1 Making DNA Origami

Making DNA origami

DNA origami recipe

We designed DNA origami by caDNAno2, software for designing 2D and 3D DNA origami.
Our DNA origami has 141 staples that have 30nt free single-stranded parts outside the DNA origami. The sequence of the parts is “each DNA origami staple-TTTTTTTTTTTTTTTCTGTCGCATCGAGAG”.
Between the staple and unique (CTGTCGCATCGAGAG) sequences, 15 T bases are inserted. They are to make a T loop. Thanks to this T loop, single-stranded DNA complementary to the unique sequences (such as Origami-anchor DNA) are expected to easily hybridize with the unique sequence.
The 30nt single-stranded parts are stable till 37 degrees, according to NUPACK).
The 141 staples have the same length so that they may be present at the same intervals in the DNA origami.
Each side of our origami is not fully covered with staples, and single-stranded M13 remains. This is for preventing π-π interaction and stacking by hydrophobic interaction between base pairs of double-stranded DNA.
This design enables each DNA origami to exist individually.

The list of strands

The other strands exept DNA origami staples used in our experiment are shown in Table2.
The sequence of Origami-anchor DNA is shown below (at the first sequence in Table2). For labeling, we also attached fluorescent-tagged DNA (at the second in Table2) to our DNA origami.
To hybridize both Origami-anchor DNA and fluorescent-tagged DNA with the same unique single-stranded parts of our Origami, we arranged two kinds of adaptor DNA (at the third and fourth in Table2). One adaptor has complementary sequences to both the unique sequence and Origami-anchor DNA. The other has complementary sequences to both the unique sequence and the fluorescent-tagged DNA. Thanks to these two adaptors, two different strands can bind to the same unique sequence.

The kinds of DNAtrands

Its sequence

Origami-anchor DNA

CCAGAAGACG

Fluorescent-tagged DNA

ACTAGTGAGTGCAGCAGTCGTACCA

Adaptor strand for Origami-anchor DNA and the unique sequence in DNA origami

CGTCTTCTGGCTCTCGATGCGACAG

Adaptor strand for fluorescent-tagged DNA and the unique sequence in DNA origami

TGGTACGACTGCTGCACTCACTAGTCTCTCGATGCGACAG

Table2 The sequence of the strands

Annealing of DNA origami

The annealing solution is shown in Table3. The annealing was conducted for 2 hours and 51minutes (from 95 to 25 degrees: lower 1 degree per 2 minutes).

Annealing solution with fluorescent-tagged DNA 50µl

84nM M13mp18

2.38µl

Staples

1µM migihaji

1µl

1µM hidarihaji

1µl

1µM ashibatemae

1µl

200nM ashiba

5µl

1µM cholesterol-hybridizing ssDNA

3µl

1µM fluorescent-tagged DNA-hybridizing ssDNA

3µl

5xTAE Mg2+

10µl

mQ

20.62µl

1µM fluorescent-tagged DNA

3µM

Table3 Annealing solution with fluorescent-tagged DNA

Annealing solution with no fluorescent-tagged DNA (control) 50µl
We changed 3µl fluorescent-tagged DNA in the above solution into the same quantity of mQ.

2-1-2 Labeling DNA Origami with fluorescent-tagged DNA

Electrophoresis

We confirmed that our DNA origami was fluorescently labeled by electrophoresis.

50µl of Annealing solution with fluorescent-tagged DNA (used in 2-1-1 Making DNA origami) contains 3µl of 1µM fluorescent-tagged DNA.
To see if the origami binds to the fluorescent-tagged DNA in shorter time, we added 0.6µl of 1µM fluorescent-tagged DNA into 10 µl control annealing solution, and left it for 40 minutes.

Agarose gel recipe: 0.4g agarose, 0.8ml 50xTAE, 39.2ml mQ

The electrophoresis was conducted with 1% agarose gel, CV 100V, for 50 minutes.

2-1-3 Disruption of liposomes by DNA Origami (microscopic analysis)

Concentration of Origami-anchor DNA

To float Origami-anchor DNA on the surface of liposome, we added Origami-anchor DNA into liposomes at the final concentration of 0.018, 0.069, 1.8, and 6.9µM. Each sample was as follows.

Observation by phase and fluorescent microscope

We observed each sample with a phase microscope.

Then we added 2µl DNA origami into each sample and saw if some change would happen with a fluorescent microscope.
The DNA origami for fluorescent microscope observation was made according to Table4 annealing solution. It contained more cholesterol-hybridizing ssDNAs and fluorescent-tagged DNA-hybridizing ssDNAs than Annealing solution used in 2-1-1, because we considered a sample with more fluorescent molecules was suitable for observation.

84nM M13mp18

2.38µl

Staples

1µM migihaji

1µl

1µM hidarihaji

1µl

1µM ashibatemae

1µl

200nM ashiba

5µl

100µM cholesterol-hybridizing ssDNA

4.23µl

100µM fluorescent-tagged DNA-hybridizing ssDNA

4.23µl

5xTAE Mg2+

10µl

mQ

23.54µl

Table4 50µl Annealing solution for fluorescent microscope observation

After annealing, we added 4.23µl 100µM fluorescent-tagged DNA (the same quantity of fluorescent-tagged DNA-hybridizing ssDNA).

2-1-4 Disruption of liposomes by DNA Origami (quantitative analysis)

Making liposome

Liposomes were formed by the droplet-transfer method (Pautot et al., PNAS, 2003).

DOPC(10mM)

20µl

DPPC(10mM)

20µl

Cholesterol(10mM)

20µl

DOPE(10mM)

20µl

chloroform

260µl

Table5 Materials for Making liposomes

1 Drying the liposomes above with argon gas and letting them stand for a night
2 Adding mineral oil 260µl to 1 and sonicating them (43Hz, 60 deg C, for 2 hours)
3 Preparing 1.5ml microtube and pouring outer buffer 50µl. Then picking up 50µl from 2 and adding it on the outer buffer (softly, to make a bilayer)

glucose(1M)

125µl

25xTAE Mg2+

10µl

mQ

110µl

Table6 Outer Buffer (250µl)

4 Preparing 0.2 ml microtube and pouring inner buffer 2µl. Then picking up 50µl from 2, adding it on the inner buffer, and mixing them by tapping

GFP(0.5 mM)

5µl

sucrose(1M)

125µl

25xTAE Mg2+

10µl

mQ

110µl

Table7 Inner Buffer (250µl)

5 Pouring all the solution (52µl) of 4 into the 1.5ml tube (softly, to make a three-layer)
6 Centrifuging it for 30 seconds and taking only the bottom layer

Disruption of liposomes by DNA Origami

Sample1 is the negative control. It is the mixture of liposome and Origami-anchor DNA.

Liposome (with GFP inside) (4mM)

10µl

Origami-anchor DNA (10uM)

25µl

1xTAE Mg2+

75µl

Table8 Sample1: negative control

Sample2 is the positive control. It is the mixture of liposome, Origami-anchor DNA, and surfactant (NP40).

Liposome (with GFP inside) (4mM)

10µl

Origami-anchor DNA (10uM)

25µl

1xTAE Mg2+

75µl

Surfactant (NP40)

2µl

Table9 Sample2: positive control

Sample3 is the mixture of liposome, Origami-anchor DNA, and Key DNA Origami.

Liposome (with GFP inside) (4mM)

10µl

Origami-anchor DNA (10uM)

25µl

1xTAE Mg2+

55µl

Key DNA (5nM)

20µl

Table10 Sample3

1. Adding Origami-anchor DNA to each sample, and leaving it for 30 minutes.
2. Adding Key DNA to each sample, and leaving it for 10 minutes.
3. Taking each sample 50µl and measuring each sample’s fluorescence intensity of 7-13 µm diameter liposomes by Cell Lab Quanta SC Flow Cytometer.

2-1-5 Confirming sequence specificity of DNA

Making liposome

We made liposomes in a spontaneous-transfer way. They were divided into two types: liposomes A of GFP, Green Fluorescent Protein, and liposomes B of Red Fluorescent Protein. These two kinds of liposomes have the same Outer Buffer but different Inner Buffer. Composition of these two buffers is as follows.